The present invention relates to derivatives of RANTES and their uses.
The protein known as RANTES was originally cloned by Schall T. J. et al., (J. Immunol. 141 1018-1025 (1988)) in Krenskyp""s laboratory at Stanford University School of Medicine. The term RANTES is derived from the phrase xe2x80x9cRaised on activation, normal T-cell derived and secretedxe2x80x9d (relevant letters underlined). Its expression is inducible by antigen stimulation or mutagen activation of T-cells. The protein is a member of the chemokine superfamily (Schall T. J., Cytokine 3 165-183 (1991); Oppenheim, J. J. et al., Ann. Rev. Immunol. 9 617-48 (1991)). The pure protein was first identified in 1992 in platelets (Kameyoshi et al., J. Exp. Med. 176 587-592 (1992)). It is a potent attractor for eosinophils, CD4+CD45RO+ T-cells, and also for monocytes. It has a sixty-eight amino acid sequence.
A receptor for RANTES has recently been cloned (Gao, J. L. et al., J. Exp. Med. 177 1421-7 (1993); Neote, K., et al., Cell 72 415-25 (1993))xe2x80x94and this has been shown to bind chemokines in the rank order of potency of MIP-1xcex1 greater than RANTES.
The present invention provides polypeptides which are antagonists of RANTES and/or of MIP-1xcex1.
Despite the considerable interest in cytokines generally and the work discussed above on RANTES and RANTES receptors in particular, prior to the present invention there has been no disclosure of the above antagonists or of the possible utilities of such antagonists.
According to the present invention there is provided a polypeptide having substantial amino acid sequence homology with RANTES and functioning as an antagonist to RANTES and/or MIP-1xcex1 in respect of one or more of the following:
(a) the chemotaxis of THP-1 cells in response to RANTES and/or in response to MIP-1xcex1;
(b) the mobilisation of calcium ions in THP-1 cells due to the presence of RANTES and/or due to the presence of MIP-1xcex1; and
(c) the binding of RANTES and/or of MIP-1xcex1 to receptors of THP-1 cells.
The polypeptides provided by the present invention are useful in further characterising RANTES and its effectsxe2x80x94for example in studying RANTES induced chemotaxis, mobilisation of calcium ions and receptor binding. They are also useful in the characterisation of the binding of RANTES to its receptors. They are useful in studying MIP-1xcex1 for corresponding reasons.
Additionally, the polypeptides of the present invention are believed to be useful in the treatment of various diseases, as will be discussed later.
A preferred polypeptide of the present invention acts as an antagonist to RANTES and/or to MIP-1xcex1 due to the presence of one or more N-terminal amino acids (which are not present at the corresponding position in RANTES and which can therefore be regarded as additional N-terminal amino acids relative to those present at the N-terminus of RANTES). These N-terminal amino acids are preferably naturally occurring (L-) amino acids (which can be incorporated by using recombinant DNA techniques or by peptide fusion techniques). However non-naturally occurring amino acids (e.g. D-amino acids) may be used. These may be incorporated by using chemical synthesis techniques.
There may be only one such additional amino acid, in which case it may be Leucine or Methionine, for example. Such polypeptides can be prepared by any suitable techniques (e.g. by using gene cloning techniques, chemical synthesis, etc.). In one embodiment of the present invention they are prepared by providing a larger polypeptide comprising a desired sequence and then using enzymatic cleavage to produce a polypeptide consisting of the desired sequence.
The polypeptides of the present invention may comprise more than one additional N-terminal amino acids e.g. they may include up to five, up to ten or up to twenty additional amino acids. In some cases over twenty additional N-terminal amino acids may be present.
Again, any suitable techniques can be used to prepare such polypeptides.
The various aspects of the present invention will now be discussed in further detail below.
The present inventors have discovered that using an E. coli expression system intended to express RANTES in a form corresponding to mature human RANTES (i.e. with the signal sequence removed) a polypeptide was expressed in which an additional N-terminal methionine was present (this was not cleaved from the remaining sequence by endogenous E. coli proteases). It was surprisingly found that the presence of this additional amino acid substantially changed the characteristics of the polypeptide relative to those of RANTES. The methionylated polypeptide (referred to herein as methionylated RANTES or Met-RANTES) was found to act as an antagonist of RANTES and of MIP-1xcex1 in various assays and was not found to have any substantial agonist activity.
It should be appreciated that in many cases where N-terminal methionylation occurs in E. coli, it makes little or no difference to a polypeptide""s properties or results in it merely having reduced levels of its previous biological activity. It was therefore totally unexpected that N-terminal methionylation would in the case of the present invention actually result in antagonistic activity.
In order to determine whether or not this effect was limited to the present of an N-terminal methionine, another polypeptide was produced, in which the N-terminal methionine was replaced with an N-terminal Leucine. Again, this was found to act as an antagonist of RANTES, as was a further polypeptide in which the N-terminal methionine was replaced with an N-terminal Glutamine.
In a preferred form, the polypeptide of the present invention has the sequence:
(i) MSPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S (sometimes referred to herein as xe2x80x9cMet-RANTESxe2x80x9d)
(ii) LSPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S (sometimes referred to herein as xe2x80x9cLeu-RANTESxe2x80x9d)
or
(iii) QSPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S (sometimes referred to herein as xe2x80x9cGln-RANTESxe2x80x9d)
or has a sequence which is substantially homologous with any of the above sequences. The polypeptide may be in a glycosylated or unglycosylated form.
The term xe2x80x9csubstantially homologousxe2x80x9d when used herein includes amino acid sequences having at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence homology with the given sequence (in order of preference). This term can include, but is not limited to, amino acid sequences having from 1 to 20, from 1 to 10 or from 1 to 5 single amino acid deletions, insertions or substitutions relative to a given sequencexe2x80x94provided that the resultant polypeptide acts as an antagonist to RANTES or to MIP-1xcex1.
The polypeptide may be in substantially pure form. It may be isolated from naturally occurring polypeptides.
It should be noted that it is well known in the art that certain amino acids can be replaced with others resulting in no substantial change in the properties of a polypeptide. Such possibilities are within the scope of the present invention.
It should also be noted that deletions or insertions of amino acids can often be made which do not substantially change the properties of a polypeptide. The present invention includes such deletions or insertions (which may be, for example up to 10, 20 or 50% of the length of the specific antagonists sequence given above). The present invention also includes within its scope fusion proteins in which the polypeptides of the present invention are fused to another moiety. This may be done, for example, for the purpose of labelling or for a medicinal purpose.
The present inventors have demonstrated that a polypeptide of the present invention can act as an antagonist to the effects of RANTES or of MIP-1xcex1 in chemotaxis, calcium mobilisation and receptor binding in THP-1 cells (a monocytic cell line). These cells are available from ATCC (American Tissue Culture Collection) and act as a good model system for studying RANTES because they show calcium responses and chemotactic responses to RANTES and MIP-1xcex1, as well as other chemokines such as MCP-1. The polypeptide can also act as an antagonist of RANTES or of MIP-1xcex1 in chemotaxis, calcium mobilisation and receptor binding in these cells. MIP-1xcex1 was originally identified as part of a Macrophage Inflammatory Polypeptide fraction (which was split into MIP-1xcex1 and -xcex2) (Obaru, K et al., J Biochem 99:885-894 (1988). Sippel, P F et al., J Immunol 142:1582-1590 (1989)). It shows chemotactic activity towards T-cells and monocytes. It has also been shown to be a potent inhibitor of stem cell proliferation.
Based upon these observations it is believed that the polypeptides of the present invention can be of utility in blocking the effects of RANTES and/or MIP-1xcex1 and can therefore be of use in therapy. A preferred use of the polypeptides of the present invention is in blocking the effects of RANTES and/or MIP-1xcex1 in the recruitment and/or activation of pro-inflammatory cells. The present invention may therefore be of utility in the treatment of diseases such as asthma, allergic rhinitis, atopic dermatitis, atheroma/atheroschleosis and rheumatoid arthritis.
In addition to the polypeptides discussed above, the present invention also covers DNA sequences coding for such polypeptides (which may be in isolated or recombinant form), vectors incorporating such sequences and host cells incorporating such vectors which are capable of expressing the polypeptides of the present invention.
The polypeptides of the present invention can be produced by expression from prokaryotic or eukaryotic host cells, utilising an appropriate DNA coding sequence. Appropriate techniques are disclosed in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Laboratory Press, USA. Alternatively, they may be produced by covalently modifying RANTES. This can be done, for example, by methionylating RANTES as its N-terminus.